In recent years, worldwide concerns about environmental issues and the depletion of limited resources have increased rapidly. As an alternative to solve such problems, the construction of production systems based on environmentally friendly and renewable organisms has been of increasing interest. Such systems can be constructed by regulating metabolic pathways in organisms to optimize metabolic fluxes for desired metabolites, and various molecular biological techniques are required in this process for regulating metabolic pathways.
Methods for regulating metabolic pathways include a method of increasing the expression of enzymes required for biosynthetic processes to enhance metabolic fluxes, and a method of preventing metabolic fluxes that are used for cell growth and the production of other metabolites and deleting genes in order to produce desired metabolites. A gene deletion method that is currently widely used is a method in which the gene to be deleted is replaced by any sequence homologous thereto using recombinase so that the function of the gene is lost (Datsenko et al, PNAS, 97(12): 6640-6645, 2000). However, this gene deletion method has the following problems.
First, the time required that is for gene deletion is relatively long. Considering the time required that is replace the chromosomal gene with the homologous sequence and the time that is required to remove an antibiotic resistance gene, used to select cells from which gene deletion was normally removed, from the chromosome, a time of one week or longer is require to remove one gene. This long gene deletion time interferes with the development of metabolic engineering focused on efficiently producing metabolites by metabolic pathway regulation, compared to molecular biology technology that has developed rapidly in recent years.
Second, gene deletion causes complete loss of function of the gene. This suggests that the expression level of the gene cannot be regulated to a desired level. In order to efficiently produce a desired metabolite in an intracellular metabolic pathway, gene deletion is attempted to block a metabolic flux that goes to the metabolic pathway. However, if the blocked metabolic flux produces a substance essential for cell growth, the growth of the cells from which the gene was deleted will be significantly inhibited or will not occur. In order to maximize the efficiency of production of a desired metabolite, cells that produce the desired metabolite should be easily grown while a metabolic flux to the desired metabolite should also be optimized. Thus, there is a need for a method capable of producing a desired substance while maintaining cell growth by regulating the level of gene expression, rather than simply deleting the gene for the purpose of regulating the metabolic flux.
Third, the gene deleted from the chromosome by a conventional gene deletion method is difficult to restore. In addition, if the deletion of the same gene is attempted in other strains, all the processes should be attempted again, and thus large amounts of time and effort are required.
Thus, in order to overcome the limitations of the conventional gene deletion method, it should be possible to reduce the expression level of the gene of interest without modifying the chromosomal sequence of the strain. Also, it should be possible to regulate the expression level of the gene and to easily apply the regulation of expression of the same gene to other strains. Further, it is required to construct and apply the gene regulatory system in a fast and convenient manner.
Accordingly, the present inventors attempted to construct a short-length customized synthetic sRNA using a method satisfying the above-described requirements.
A total of 101 E. coli sRNAs are currently known (Pichon et al., Nucleic Acids Research, DOI:10.1093/nar/gkr1141, 2011), and the mechanisms thereof are currently being studied. It has been reported to date that a large number of sRNAs form a specific secondary structure, even though they have different sequences, and that the Hfq protein recognizes and binds to the sRNAs. The functions of Hfq are to increase the half life of sRNA so that the sRNA effectively functions even in a small amount, and to assist the binding of the sRNA to the target mRNA so that the target mRNA functions quickly, and to bind RNase to the target mRNA to promote the degradation of the target mRNA to thereby more effectively reduce the expression of the gene.
The fundamental functions of E. coli sRNA have been continuously studied, but there are little or no reports on the use of the above-described mechanisms to construct synthetic sRNAs. According to recent reports on the construction of E. coli sRNAs, effective sRNAs have been constructed by random mutagenesis and screening (Sharma et al, ACS Synthetic Biology, DOI: 10.1021/sb200001q, 2011).
Accordingly, the present inventors have made extensive efforts to construct a novel, short-length customized synthetic sRNA in order to overcome the limitations of the conventional gene deletion method, and as a result, have construct a synthetic sRNA comprising an Hfq binding site, derived from MicC, SgrS and/or MicF, and a region homologous to the target mRNA such as DsRed2 mRNA, AraC mRNA, KanR mRNA or LuxR mRNA, and have found that test results for the expression of the target mRNA indicate that an efficient sRNA can be constructed without relying on conventional random methods. In addition, the present inventors have constructed synthetic sRNAs that inhibit the expression of tyrR (tyrosine regulator), csrA (carbon storage regulator), pgi (glucose-6-phosphate isomerase), ppc (phosphoenolpyruvate carboxylase) and the like, and have found that these synthetic sRNAs show effects similar to those of actual gene deletion and can be used to regulate the production of a desired metabolite, thereby completing the present invention.
The information disclosed in the Background Art section is only for the enhancement of understanding of the background of the present invention, and therefore may not contain information that forms a prior art that would already be known to a person of ordinary skill in the art.